Acute respiratory failure from acute lung injury is a major clinical problem with substantial mortality and morbidity. The overall objective of this proposal is to test the value of cell-based therapy with mesenchymal stem cells (MSC) for the treatment of experimental acute lung injury and restoring normal lung fluid balance in acute lung injury. Aim 1 is designed to determine the therapeutic effects of intrapulmonary and intravenous delivery of MSC on pulmonary edema and survival in clinically relevant mouse models of acute lung injury. Aim 2 will evaluate the mechanisms responsible for the beneficial effects of MSC treatment in the clinically relevant models of pulmonary edema from acute lung injury in mice studied in Aim 1, with a specific focus on the role of interleukin-1 receptor antagonist (IL-1ra) and interleukin-10. Aim 3 is designed to test the therapeutic and mechanistic effects of allogeneic human MSC on restoring alveolar epithelial fluid transport in an in vitro model of cultured human alveolar epithelial type II cells and in an ex vivo perfused human lung preparation. The proposed studies in Aims 1 and 2 will advance our understanding of the mechanisms by which MSCs reduce lung injury and pulmonary edema as well as advance our knowledge of the biology of MSCs in the setting of acute lung injury in mice. Aim 3 will translate these findings to the human lung by using cultured monolayers of polarized human alveolar epithelial type II cells and an ex vivo perfused human lung preparation. The results of these experiments will help make it possible to determine whether testing MSCs in patients with acute lung injury is appropriate. The proposed work will also uncover mechanisms by which MSC reduces pulmonary edema from acute lung injury, and these insights will advance the fields of lung fluid balance, pulmonary edema, and acute lung injury. PROJECT NARRATIVE. This research proposal has direct relevance to the clinical problem of acute respiratory failure from pulmonary edema secondary to acute lung injury, a cause of morbidity and mortality in the United States. The proposal will test novel strategies to use cell based therapy for the treatment of experimental acute lung injury including mouse models of lung injury as well as innovative models that use human lung epithelial cells and intact human lungs harvested from brain dead subjects.